Cereulide Production Research Articles

The megaplasmid pCER270 of Bacillus cereus emetic strain affects the timing of the sporulation process, spore resistance properties, and germination.

The acquisition of new mobile genetic elements, such as plasmids, is essential for the pathogenesis and adaptation of bacteria belonging to the Bacillus cereus group. This can confer new phenotypic traits and beneficial functions that enable bacteria to adapt to changing environments and colonize new ecological niches. Emetic B. cereus strains cause food poisoning linked to the production of cereulide, the emetic toxin whose synthesis is due to the presence of plasmid pCER270. In the environment, cereulide provides a competitive advantage in producing bacteria against various competitors or predators. This study demonstrates that pCER270 also regulates the sporulation process, resulting in spores with improved heat resistance and germination capacity. The transfer of plasmid pCER270 among different strains of the B. cereus group may enhance their adaptation to new environments. This raises the question of the emergence of new pathogenic strains, which could pose a serious threat to human health.

Characterization of emetic Bacillus cereus biofilm formation and cereulide production in biofilm

Bacillus cereus is a well-known foodborne pathogen that can cause human diseases, including vomiting caused by emetic toxin, cereulide, requiring 105-108 cells per gram to cause the disease. The bacterial cells may be eliminated during processing, but cereulide can survive in most processing techniques due to its resistance to high temperatures, extreme pH and proteolytic enzymes. Herein, we reported dynamic processes of biofilm formation of four different types and cereulide production within the biofilm. Confocal laser scanning microscopy (CLSM) images revealed that biofilms of the four different types reach each stage at different time points. Among the extracellular polymeric substances (EPS) components of the four biofilms formed by the emetic B. cereus F4810/72 strain, proteins account for the majority. In addition, there are significant differences (p < 0.05) in the EPS components at the same stage among biofilms of different types. The time point at which cereulide was first detected in the four types of biofilms was 24 h. In the biofilm of B. cereus formed in ultra-high-temperature (UHT) milk, the first peak of cereulide appeared at 72 h. The cereulide content of the biofilms formed in BHI was mostly higher than that of the biofilms formed in UHT milk. This study contributes to a better understanding of food safety issues in the industry caused by biofilm and cereulide toxin produced by B. cereus.

Cereulide production capacities and genetic properties of 31 emetic Bacillus cereus group strains

The highly potent toxin cereulide is a frequent cause of foodborne intoxications. This extremely resistant toxin is produced by Bacillus cereus group strains carrying the plasmid encoded cesHPTABCD gene cluster. It is known that the capacities to produce cereulide vary greatly between different strains but the genetic background of these variations is not clear.In this study, cereulide production capacities were associated with genetic characteristics. For this, cereulide levels in cultures of 31 strains were determined after incubation in tryptic soy broth for 24 h at 24 °C, 30 °C and 37 °C. Whole genome sequencing based data were used for an in-depth characterization of gene sequences related to cereulide production. The taxonomy, population structure and phylogenetic relationships of the strains were evaluated based on average nucleotide identity, multi-locus sequence typing (MLST), core genome MLST and single nucleotide polymorphism analyses. Despite a limited strain number, the approach of a genome wide association study (GWAS) was tested to link genetic variation with cereulide quantities.Our study confirms strain-dependent differences in cereulide production. For most strains, these differences were not explainable by sequence variations in the cesHPTABCD gene cluster or the regulatory genes abrB, spo0A, codY and pagRBc. Likewise, the population structure and phylogeny of the tested strains did not comprehensively reflect the cereulide production capacities. GWAS yielded first hints for associated proteins, while their possible effect on cereulide synthesis remains to be further investigated.

Effect of Temperature, pH, and aw on Cereulide Synthesis and Regulator Genes Transcription with Respect to Bacillus cereus Growth and Cereulide Production.

Bacillus cereus is a food-borne pathogen that can produce cereulide in the growth period, which causes food poisoning symptoms. Due to its resistance to heat, extreme pH, and proteolytic enzymes, cereulide poses a serious threat to food safety. Temperature, pH, and aw can influence cereulide production, but there is still a lack of research with multi-environmental impacts. In this study, the effects of temperature (15~45 °C), pH (5~8), and aw (0.945~0.996) on the emetic reference strain B. cereus F4810/72 growth, cereulide production, relevant ces genes (cesA, cesB, cesP), and transcription regulators genes (codY and abrB) expression at transcription level were studied. B. cereus survived for 4~53 h or grew to 6.85~8.15 log10 CFU/mL in environmental combinations. Cereulide accumulation was higher in mid-temperature, acidic, or high aw environments. Increased temperature resulted in a lower cereulide concentration at pH 8 or aw of 0.970. The lowest cereulide concentration was found at pH 6.5 with an increased aw from 0.970 to 0.996. Water activity had a strong effect on transcriptional regulator genes as well as the cesB gene, and temperature was the main effect factor of cesP gene expression. Moreover, environmental factors also impact cereulide synthesis at transcriptional levels thereby altering the cereulide concentrations. The interaction of environmental factors may result in the survival of B. cereus without growth for a period. Gene expression is affected by environmental factors, and temperature and pH may be the main factors influencing the correlation between B. cereus growth and cereulide formation. This study contributed to an initial understanding of the intrinsic link between the impact of environmental factors and cereulide formation and provided valuable information for clarifying the mechanism of cereulide synthesis in combined environmental conditions.

The potential of lactose to inhibit cereulide biosynthesis of emetic Bacillus cereus in milk

This study aims to investigate the potential role of lactose on cereulide biosynthesis by emetic Bacillus cereus in dairy matrices. The cereulide yields in whole milk and lactose-free milk were investigated using the emetic reference strain F4810/72. To eliminate the influence of complex food substrates, the LB medium model was further used to characterize the effect of lactose on cereulide produced by F4810/72 and five other emetic B. cereus strains. Results showed that the lactose-free milk displayed a 13-fold higher amount of cereulide than whole milk, but the cereulide level could be reduced by 91 % when the lactose content was restored. The significant inhibition of lactose on cereulide yields of all tested B. cereus strains was observed in LB medium, showing a dose-dependent manner with inhibition rates ranging of 89–98 %. The growth curves and lactose utilization patterns of all strains demonstrated that B. cereus cannot utilize lactose as a carbon source and lactose might act as a signal molecule to regulate cereulide production. Moreover, lactose strongly repressed the expression of cereulide synthetase genes (ces), possibly by inhibiting the key regulator Spo0A at the transcriptional level. Our findings highlight the potential of lactose as an effective strategy to control cereulide production in food.

Get to Know Your Neighbors: Characterization of Close Bacillus anthracis Isolates and Toxin Profile Diversity in the Bacillus cereus Group.

Unexpected atypical isolates of Bacillus cereus s.l. occasionally challenge conventional microbiology and even the most advanced techniques for anthrax detection. For anticipating and gaining trust, 65 isolates of Bacillus cereus s.l. of diverse origin were sequenced and characterized. The BTyper3 tool was used for assignation to genomospecies B. mosaicus (34), B. cereus s.s (29) and B. toyonensis (2), as well as virulence factors and toxin profiling. None of them carried any capsule or anthrax-toxin genes. All harbored the non-hemolytic toxin nheABC and sphygomyelinase spH genes, whereas 41 (63%), 30 (46%), 11 (17%) and 6 (9%) isolates harbored cytK-2, hblABCD, cesABCD and at least one insecticidal toxin gene, respectively. Matrix-assisted laser desorption ionization-time of flight mass spectrometry confirmed the production of cereulide (ces genes). Phylogeny inferred from single-nucleotide polymorphisms positioned isolates relative to the B. anthracis lineage. One isolate (BC38B) was of particular interest as it appeared to be the closest B. anthracis neighbor described so far. It harbored a large plasmid similar to other previously described B. cereus s.l. megaplasmids and at a lower extent to pXO1. Whereas bacterial collection is enriched, these high-quality public genetic data offer additional knowledge for better risk assessment using future NGS-based technologies of detection.

Role of Bacillus cereus biofilm formation behavior in virulence and pathogenic characteristics

AbstractThis study aimed to examine the potential link between Bacillus cereus biofilm formation, virulence, and pathogenicity. The biofilm formation abilities of nine B. cereus strains isolated from food and two reference strains (ATCC 10876 and ATCC 25621) were measured using a crystal violet assay. Among the tested strains, three strains (GIHE 617‐5, GIHE 86‐09, and GIHE 728‐17) and both reference strains were capable of biofilm formation. A positive correlation was obtained for higher cell surface hydrophobicity and increased biofilm formation. In contrast, HPLC analysis for elevated autoinducer‐2 (Al‐2) production revealed a negative impact on biofilm formation. PCR data indicated that all tested strains were capable of producing common B. cereus enterotoxins, including Hbl—A, C, and D, CytK, Nhe—B and C, EntFM, and BceT, but were negative for production of the emetic toxin cereulide and the pore‐forming toxin Hly II. Meanwhile, RT‐PCR data revealed a close correlation between high biofilm formation and the upregulation of several tested virulence genes for selected strains. However, elevated upregulation of virulence genes was not consistent in all of the higher biofilm‐forming strains. Cytotoxicity analysis revealed higher virulence characteristics compared to those of low biofilm‐forming strains.

Distribution of cereulide-producing Bacillus cereus in raw milk in Hokkaido, Japan, and evaluation of cereulide production

It is important to understand the distribution of strains of Bacillus cereus that produce the emetic toxin, cereulide, in raw milk, and to evaluate their impact on the safety of milk and dairy products. The distribution of cereulide-producing B. cereus was investigated for isolates from raw milk in Hokkaido, Japan, and the ability of strains to produce cereulide in milk was also studied. Only one of 393 isolates had the cereulide-producing gene, indicating very low presence of cereulide-producing strains in milk in Hokkaido. This isolate produced cereulide in milk stored at 12 and 30 °C, but not at 10 °C. It was concluded that keeping storage temperatures below 10 °C reduces the possibility of accumulation of cereulide in milk and dairy products pasteurised at temperatures at which B. cereus spores survive. Hence the possibility of occurrence of cereulide-associated vomiting by ingestion of dairy products of Hokkaido, is very low.

The Sources of Bacillus cereus Contamination and their Association with Cereulide Production in Dairy and Cooked Rice Processing Lines

Abstract Bacillus cereus, a spore-forming bacterium and frequent cause of food poisoning, poses a safety threat to dairy and rice industries due to its high contamination rates and ability to produce toxins such as cereulide. Because of widespread presence and thermal resistance of the spores, B. cereus cannot be eliminated from the environment and may survive in processing plants. Surviving spores can develop into vegetative cells, leading to a heightened risk of cereulide production in the processing environment. Both spores and vegetative cells have the ability to adhere to the surfaces of dairy plants and form biofilms, serving as the site for cereulide production and accumulation. Therefore, it is crucial for the food industry to address potential sources and pathways of B. cereus contamination and their connections to cereulide production in processing lines. In this review, sources of contamination of B. cereus, including spores, vegetative cells, and biofilms and their potential role in cereulide production at each stage of dairy and cooked rice processing were analyzed. In addition, control methods to prevent B. cereus contamination and cereulide production in processing lines were proposed, offering valuable insights for improving microbial risk management in the food industry.

A Flagella Hook Coding Gene flgE Positively Affects Biofilm Formation and Cereulide Production in Emetic Bacillus cereus.

Bacillus cereus, an important foodborne pathogen, poses a risk to food safety and quality. Robust biofilm formation ability is one of the key properties that is responsible for the food contamination and food poisoning caused by B. cereus, especially the emetic strains. To investigate the mechanism of biofilm formation in emetic B. cereus strains, we screened for the mutants that fail to form biofilms by using random mutagenesis toward B. cereus 892-1, an emetic strain with strong biofilm formation ability. When knocking out flgE, a flagellar hook encoding gene, the mutant showed disappearance of flagellar structure and swimming ability. Further analysis revealed that both pellicle and ring presented defects in the null mutant compared with the wild-type and complementary strains. Compared with the flagellar paralytic strains Δ motA and Δ motB, the inhibition of biofilm formation by Δ flgE is not only caused by the inhibition of motility. Interestingly, Δ flgE also decreased the synthesis of cereulide. To our knowledge, this is the first report showing that a flagellar component can both affect the biofilm formation and cereulide production in emetic B. cereus, which can be used as the target to control the biohazard of emetic B. cereus.

Influence of the Phagemid PfNC7401 on Cereulide-Producing Bacillus cereus NC7401.

A phagemid-cured strain, NC7401-∆Pf, was constructed to survey the biological function of the plasmidal prophage PfNC7401 in cereulide-producing Bacillus cereus NC7401. The transcriptome analysis between the mutant and the wild strains revealed a series of differentially expressed genes mainly involved in different function classifications, including the two-component signal transduction system, bacterial structure, transporters, related antibiotic response, purine biosynthesis, non-ribosomal peptide synthetases (NRPS) and related secondary metabolites, and aromatic or other amino acid synthesis. BIOLOG and phenotypic experiment analyses confirmed that PfNC7401 may affect phage immunity and the metabolism of several amino acids, including L-Alanine, which was suggested to be related to one precursor (D-Alanine) of cereulide synthesis. However, neither the transcription levels of the cereulide production-related genes (e.g., ilvB, cesA, cesB, and cesH) nor the cereulide production nor cell cytotoxicity were affected by the presence or absence of PfNC7401, corresponding with the transcriptome data, in which only four genes unrelated to cereulide synthesis on the plasmid-carrying ces gene cluster were affected by the curing of PfNC7401.

Predicting B. cereus growth and cereulide production in dairy mix

This study aims to quantify growth and cereulide production by Bacillus cereus and their potential correlation in an intermediate dairy wet-mix. Systematic experiments were carried out using the emetic reference strain F4810/72 in the suboptimal range of temperature of 12 °C to 20 °C. Growth and cereulide kinetic parameters were estimated and the three parameters (i) time to first cereulide quantification (tcer), (ii) maximum specific growth rates (μmax) and (iii) cereulide production rates (k) were modelled as a function of temperature. As temperature increased, growth lag time and tcer were shorter while microbial increase and cereulide production happened earlier, and at higher rates. Maximum concentration of cells and maximum cereulide concentration proved to be temperature-independent, reaching the average values of 7.9 ± 0.3 log10(CFU/mL) and 2.6 ± 0.2 log10(ng.g−1) respectively. Moreover, the time to reach the widely used threshold of 5 log10CFU/mL (t5log) was tested against tcer, and this suggested that this threshold can be used with increased confidence at lower temperatures to assure toxin is not quantified in this matrix. The average tcer were equal to 314 h, 118 h, 73 h and 45 h for 12 °C, 15 °C, 18 °C and 20 °C respectively. A validation study was performed using independent data sets obtained with the same strain in other dairy matrices. The microbial growth models presented good predictive power even when extrapolated beyond the temperature range of construction. Nevertheless, the models proposed for prediction of toxin production over time presented limitations, especially for food matrices that deviate significantly from the original matrix for which the model was developed, making cereulide predictions less accurate. Our findings suggest that similar modelling approaches can be used to predict growth, time to first cereulide quantification as well as cereulide formation over time for a specific matrix, but that matrix-extrapolations are more suitable for growth than for cereulide.

Impact of Phytochemicals on Viability and Cereulide Toxin Synthesis in Bacillus cereus Revealed by a Novel High-Throughput Method, Coupling an AlamarBlue-Based Assay with UPLC-MS/MS.

Due to its food-poisoning potential, Bacillus cereus has attracted the attention of the food industry. The cereulide-toxin-producing subgroup is of particular concern, as cereulide toxin is implicated in broadscale food-borne outbreaks and occasionally causes fatalities. The health risks associated with long-term cereulide exposure at low doses remain largely unexplored. Natural substances, such as plant-based secondary metabolites, are widely known for their effective antibacterial potential, which makes them promising as ingredients in food and also as a surrogate for antibiotics. In this work, we tested a range of structurally related phytochemicals, including benzene derivatives, monoterpenes, hydroxycinnamic acid derivatives and vitamins, for their inhibitory effects on the growth of B. cereus and the production of cereulide toxin. For this purpose, we developed a high-throughput, small-scale method which allowed us to analyze B. cereus survival and cereulide production simultaneously in one workflow by coupling an AlamarBlue-based viability assay with ultraperformance liquid chromatography–mass spectrometry (UPLC-MS/MS). This combinatory method allowed us to identify not only phytochemicals with high antibacterial potential, but also ones specifically eradicating cereulide biosynthesis already at very low concentrations, such as gingerol and curcumin.

Prevalence of Cereulide-Producing Bacillus cereus in Pasteurized Milk

To recognize the risk of Bacillus cereus in pasteurized milk, we investigated the prevalence of B. cereus and the rate of the production of cereulide from B. cereus isolates. B. cereus was found in 66 out of 101 (65.3%) domestically pasteurized milk samples in Japan. The ces gene was identified in 3 out of 90 B. cereus isolates that were isolated from three samples (one product) among the 101 samples. The ces gene positive isolate, the reference strain F4810/72 and a B. cereus isolate collected in a food poisoning incident were shown the productivity of cereulide using an LC-MS/MS analysis. The LC-MS/MS analysis was confirmed the ability of identification and quantification of cereulide produced in the milk samples. In this study, it was shown that B. cereus strains are prevalent in pasteurized milk, some of these strains produce cereulide, and confirmed usefulness of LC-MS/MS analysis to detect cereulide in milk.

Evaluation of growth and cereulide production by Bacillus cereus isolated from cooked rice

Conditions influencing Bacillus cereus growth and cereulide production, such as temperature and pH, were evaluated at varying incubation periods. The growth and cereulide production at different temperatures and pH values ranging from 10 to 40 ºC and 5.0 to 8.5, respectively showed that the temperature from 20 to 30 ºC and at pH from 6.0 to 7.0 gave the optimum growth and cereulide production by B. cereus SA105. pH below 6.0 resulted in reduced growth and cereulide production. Cereulide production increased along with the incubation period, and maximum cereulide titre (ng/mL) of 1219.1±8.90 was obtained after 6 days of incubation at 30 ºC and pH 6.5 under static conditions. There was no quantifiable toxin at incubation temperatures of 10 and 40 ºC by B. cereus SA105. This work further reveals that B. cereus growth and cereulide production was significantly affected by temperature and pH in relation to the incubation period. Furthermore, the findings of this study will serve as a means for reducing the diversity of emetic toxin-producing B. cereus population in food and food products, thus preventing food poisoning.

Evaluation of growth and cereulide production by Bacillus cereus isolated from cooked rice

Conditions influencing Bacillus cereus growth and cereulide production, such as temperature and pH, were evaluated at varying incubation periods. The growth and cereulide production at different temperatures and pH values ranging from 10 to 40 ºC and 5.0 to 8.5, respectively showed that the temperature from 20 to 30 ºC and at pH from 6.0 to 7.0 gave the optimum growth and cereulide production by B. cereus SA105. pH below 6.0 resulted in reduced growth and cereulide production. Cereulide production increased along with the incubation period, and maximum cereulide titre (ng/mL) of 1219.1±8.90 was obtained after 6 days of incubation at 30 ºC and pH 6.5 under static conditions. There was no quantifiable toxin at incubation temperatures of 10 and 40 ºC by B. cereus SA105. This work further reveals that B. cereus growth and cereulide production was significantly affected by temperature and pH in relation to the incubation period. Furthermore, the findings of this study will serve as a means for reducing the diversity of emetic toxin-producing B. cereus population in food and food products, thus preventing food poisoning.

Chemical structure of hydrolysates of cereulide and their time course profile

Hydrolysates of an emetic toxin cereulide were found in the broth of Bacillus cereus. The ester cleaved depsipeptides of cereulide were synthesized using liquid phase fragment condensation method starting from commercially available amino acids. The chemical structure of hydrolysates was verified tetradepsipeptide l-O-Val-l-Val-D-O-Leu-d-Ala and dodecadepsipeptide (D-O-Leu-d-Ala-l-O-Val-l-Val)3 using LC-TOFMS. Quantitative analysis of cereulide in the broth revealed production of cereulide in the stationary phase and decomposition in the death phase. The increase in tetradepsipeptide continued after the stationary phase until decomposition occurred.

Identification of the Gene Responsible for Cereulide Production of Bacillus Cereus in Some Meat Products

This study aimed to examine the quality and safety of some street vended meat meals in Alexandria governorate, Egypt. 105 sample of meat meals including 35 samples each of Kofta, pasta and Hawawshi were randomly collected from the street mainly for Bacillus Cereus isolation and detection of their virulent gene responsible for Cereulide production by PCR technique. As well as total aerobic count and total anaerobic count were done to all samples to evaluate the hygienic condition of these products.. Bacillus cereus was detected in 45(42.9%) samples out of 105 examined meat meal samples, and the Mean ± SEM of B. cereus in Kofta, Pasta & Hawawshi were 5.70 x 102± 1.39 x 102, 2.37 x 102± 0.43 x 102 and 5.10 x 102± 0.69 x 102 respectively. theCereulide gene coud be detected in some isolates from meat meals samples which were 3 isolates from each product and it was negative in all isolates from Kofta (0/3), while in Pasta two isolates from the 3 tested ones were positive to Cereulide virulent gene(2/3), and in Hawawshi all the tested isolates were positive to Cereulide virulent gene(3/3). The mean value of total aerobic count in Kofta, Pasta &Hawawshi were 4.55 x 104± 0.17 x 104, 5.07 x 103± 0.11 x 103 & 1.21 x 104± 0.27 x 104 respectively. At the same time we had an unneglectable results from the anaerobic samples analysis which had been recorded where the Mean ± SEM of Kofta, Pasta &Hawawshi were 4.54 x 10 ± 0.68 x 10 ,9.29 x 10 ± 1.90 x 10, 6.87 x 10 ± 0.97 x 10 respectively. Recommendation and measures that should be implemented to introduce safe meat meals were discussed.

CesH Represses Cereulide Synthesis as an Alpha/Beta Fold Hydrolase in Bacillus cereus.

Cereulide is notorious as a heat-stable emetic toxin produced by Bacillus cereus and glucose is supposed to be an ingredient supporting its formation. This study showed that glucose addition benefited on cell growth and the early transcription of genes involved in substrate accumulation and toxin synthesis, but it played a negative role in the final production of cereulide. Meanwhile, a lasting enhancement of cesH transcription was observed with the addition of glucose. Moreover, the cereulide production in ΔcesH was obviously higher than that in the wild type. This indicates that CesH has a repression effect on cereulide production. Bioinformatics analysis revealed that CesH was an alpha/beta hydrolase that probably associated with the cell membrane, which was verified by subcellular localization. The esterase activity against para-nitrophenyl acetate (PNPC2) of the recombinant CesH was confirmed. Although no sign of ester bond cleavage in cereulide or valinomycin was demonstrated in in vitro assays, CesH could reverse the cereulide analogue sensitivity of Bacillus subtilis in vivo, by which toxin degradation was facilitated. Moreover, site directed mutations identified that the conserved catalytic triad of CesH might consist of Serine 86, Glutamate 199, and Histidine 227. These results help us to understand the regulation of cereulide production and provide clues for developing control measurements.

Elaboration and validation of the method for the quantification of the emetic toxin of Bacillus cereus as described in EN-ISO 18465 - Microbiology of the food chain – Quantitative determination of emetic toxin (cereulide) using LC-MS/MS

A method for the quantification of the Bacillus cereus emetic toxin (cereulide) was developed and validated. The method principle is based on LC-MS as this is the most sensitive and specific method for cereulide. Therefore the study design is different from the microbiological methods validated under this mandate. As the method had to be developed a two stage validation study approach was used. The first stage (pre-study) focussed on the method applicability and the experience of the laboratories with the method. Based on the outcome of the pre-study and comments received during voting at CEN and ISO level a final method was agreed to be used for the second stage the (final) validation of the method. In the final (validation) study samples of cooked rice (both artificially contaminated with cereulide or contaminated with B. cereus for production of cereulide in the rice) and 6 other food matrices (fried rice dish, cream pastry with chocolate, hotdog sausage, mini pancakes, vanilla custard and infant formula) were used. All these samples were spiked by the participating laboratories using standard solutions of cereulide supplied by the organising laboratory. The results of the study indicate that the method is fit for purpose. Repeatability values were obtained of 0.6 μg/kg at low level spike (ca. 5 μg/kg) and 7 to 9.6 μg/kg at high level spike (ca. 75 μg/kg). Reproducibility at low spike level ranged from 0.6 to 0.9 μg/kg and from 8.7 to 14.5 μg/kg at high spike level. Recovery from the spiked samples ranged between 96.5% for mini-pancakes to 99.3% for fries rice dish.